A Molecular Phylogenetic Assessment of Pseudendoclonium Richard F

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Masters Theses 1911 - February 2014

2007 A Molecular Phylogenetic Assessment of Pseudendoclonium Richard F. Mullins University of Massachusetts Amherst

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A MOLECULAR PHYLOGENETIC ASSESSMENT OF PSEUDENDOCLONIUM

A Thesis Presented

RICHARD F MULLINS, JR.

Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

September 2007

Plant Biology Graduate Program A MOLECULAR PHYLOGENETIC ASSESSMENT OF PSEUDENDOCLONIUM

A Thesis Presented

RICHARD F. MULLINS, JR.

Approved as to style and content by:

______Daniel R. Cooley, Chair

______Robert L. Wick, Member

______Margaret A. Riley, Member

______Elsbeth L. Walker, Director Plant Biology Graduate Program ACKNOWLEDGMENTS

It is with deepest gratitude that I acknowledge the invaluable assistance and untiring support of my good friend and mentor Dr. Robert T. Wilce. Completion of this project would not have been possible without Bob’s persistent encouragement and insightful advice. His kindness and generosity have been of such magnitude that “thank you” doesn’t even begin to cover it.

I thank Dr. Daniel R. Cooley for sharing his extensive practical and professional wisdom as my academic advisor. I am especially grateful to Dan for his efforts securing financial support for my studies and for laboratory supplies.

I am grateful to Dr. Robert L. Wick for his participation as a thesis committee member and for his helpful advice. I thank Rob also for a solid introduction to the science of Plant Pathology, for providing teaching assistantships and for sharing laboratory equipment.

I am fortunate to have Dr. Margaret A. Riley as a thesis committee member and as a teacher. I could not have effected the phylogenetic analyses that are of central importance to this thesis, without having taken her course in molecular evolution. I am most grateful that she has taken time from a very busy schedule to review this work.

N. Ezekiel Nims has and deserves my sincere gratitude for his assistance with laboratory protocol. Zeke rescued me from the PCR doldrums.

Milind Chavan has been most helpful with the details of PCR. I thank him for his time and the patience required in answering the countless questions of a novice.

I thank the Plant Biology Graduate Program for their accommodation of a “non- traditional” student and for the opportunity to pursue a graduate degree.

iii From my perspective, Susan Capistran is the most dedicated program director on this campus. Thank you so much for your assistance, patience and understanding.

David Fill, a maintenance supervisor for Umass Dining Services, has provided me with part-time employment for the duration of my graduate career. As my boss,

Dave has been most understanding and supportive of my graduate studies. He has patiently allowed scheduling changes necessary for the completion of this work. For this, I owe him much and offer my sincere gratitude.

ABSTRACT

A MOLECULAR PHYLOGENETIC ASSESSMENT OF PSEUDENDOCLONIUM

SEPTEMBER 2007

RICHARD F. MULLINS JR., B.S., UNIVERSITY OF MASSACHUSETTS AMHERST

Directed by: Professor Daniel R. Cooley

Pseudendoclonium was established in 1900 by N. Wille to include a crust- forming green microalga occurring near the high water line on jetties in Drobak,

Norway. Ordinal and familial affiliation of the genus have remained uncertain due to a lack of distinguishing morphological characteristics and because molecular phylogenetic data have not been generated for the type species. Ribosomal SSU rDNA sequence data for Pseudendoclonium submarinum, the type species, are presented.

Phylogenetic analysis of these data place Pseudendoclonium within the Ulvales. SSU rDNA sequence data from three additional species, Pseudendoclonium basiliense,

Pseudendoclonium akinetum and Pseudendoclonium fucicola are included in the analyses and clearly support the hypothesis that Pseudendoclonium is polyphyletic.

Based on the sequence data, P. submarinum and P. fucicola share ulvalean lineage, but these algae are not congeneric and P. fucicola must be removed from

Pseudendoclonium. Sequence data support the classification of P. basiliense and P. akinetum as distinct species of a single genus. The close affiliation of these two species with Ulothrix and other Ulotrichalean genera, however, reveals their ordinal separation from P. submarinum. P. basiliense and P. akinetum must also be removed from

Pseudendoclonium and require generic reassignment within the Ulotrichales.

v TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... iii

ABSTRACT...... v

LIST OF TABLES...... viii

LIST OF FIGURES ...... ix

CHAPTER

1. THE GENUS PSEUDENDOCLONIUM ...... 1

1.1 Introduction...... 1 1.2 Taxonomic History...... 2

2. MATERIALS AND METHODS...... 6

2.1 Isolation and Culture...... 6 2.2 Microscopy ...... 7 2.3 DNA Extraction...... 8 2.4 DNA Amplification ...... 9

2.4.1 PCR Primers...... 9

2.5 Detection and Analysis...... 10

2.5.1 Purification...... 10 2.5.2 Sequencing...... 10 2.5.3 Sequencing Primers...... 11 2.5.4 Sequence Editing...... 11

2.6 Phylogenetic Analysis...... 12

3. RESULTS...... 15

3.1 General...... 15 3.2 Partial-Sequence Analysis ...... 16 3.3 Full-Sequence Analysis ...... 18

4. DISCUSSION...... 20

4.1 Generic...... 20 4.2 Specific ...... 21

5. CONCLUSION...... 27

5.1 Evolutionary Considerations...... 27 5.2 Specific Assignment ...... 28

REFERENCES ...... 38

vii LIST OF TABLES

Table page

1.1 The Genus Pseudendoclonium ...... 32

2.1 Composition of Modified von Stosch’s Enriched Seawater Medium ...... 32

2.2 Oligonucleotide Primers Used in this Study...... 33

3.1 SSU rDNA Sequence of Pseudendoclonium submarinum ...... 34

viii LIST OF FIGURES

Figure page

1.1 Growth Habit of P. submarinum in Liquid Culture...... 35

1.2 Scanning Electron Micrographs of Pseudendoclonium submarinum ...... 35

3.1 Maximum Likelihood Phylogram Inferred from Partial 18s rDNA Sequences of 25 Green Algal Taxa Illustrating Ordinal Affiliation of Four Species of Pseudendoclonium as the Genus is Currently Circumscribed...... 36

3.2 Maximum Likelihood Phylogram Inferred from 18S rDNA Sequences of 22 Green Algal Taxa Illustrating Ordinal Affiliation of Three Species of Pseudendoclonium as the Genus is Currently Circumscribed ...... 37

CHAPTER 1

THE GENUS PSEUDENDOCLONIUM

1.1 Introduction

Pseudendoclonium was established by N. Wille (1900) to accommodate a crust- forming green alga that he recognized near the high water line on jetties in Drobak,

Norway. This microalgal species was described as having short, prostrate and erect irregularly branched filaments, and cells containing a single, parietal chloroplast with one pyrenoid. Wille also described rhizoid-like cells of diminished chlorophyll content presumed to assist attachment of the prostrate basal disc. Wille noticed that sections of the alga easily separated into what he termed Pleuroccocus -like fragments and considered this a distinguishing feature. Pseudendoclonium submarinum Wille remains the type species of the genus.

Other details of vegetative and reproductive morphology are presented in

Wille’s original manuscript. Asexual reproduction is accomplished either by the production of ovoid, quadriflagellate zoospores or by akinetes. Akinetes are described as being of two types: thick-walled resting cells and thin-walled cells that germinate without a dormant period. Sexual reproduction was not observed by Wille or subsequent investigators of the species.

In liquid culture the mature thallus appears as a tuft of highly-branched short filaments arising from the basal disc (Fig.1.1, Fig. 1.2). Tufts are thus roughly circular in outline when viewed from above. They are more or less hemispherical in vertical section as longer and more highly branched filaments arise from the center of the

thallus. Branching is highly irregular and without any distinct pattern or apparent organization. Margins are uneven due to the haphazard protrusion of filaments. The alga attaches to any firm substratum. It eventually forms a continuous crust as new tufts arise from zoospores or akinetes and develop between or upon the older thalli. Individual mature tufts measure 0.5 to 1.0 mm in diameter. Organisms several years in age attained a maximum diameter of 1.6 mm.

During the past century eight additional species have been referred to

Pseudendoclonium on the basis of morphological and reproductive features. A complete list of species is given (Table 1.1).

The following systematic history of Pseudendoclonium illustrates the uncertainty encountered in the use of classical morphological characteristics to reconstruct phylogeny.

1.2 Taxonomic History

Familial association of Pseudendoclonium with the Chaetophoraceae originated with Wille. The new alga was placed in this family primarily because of its much- branched heterotrichous growth habit. Wille considered P. submarinum to be a highly reduced and adapted form descended from the upright and highly branched

Stigeoclonium and Endocladium (Wille 1900).

Collins (1909) was first to describe the occurrence of Pseudendoclonium submarinum in North America from the coasts of three New England states and retained its familial placement in the Chaetophoraceae. In a broad study of green algae, Collins proposed a system of six orders within the class Chlorophyceae. His very broadly circumscribed order Ulotrichales included virtually all branched and unbranched

filamentous green algae from freshwater and marine habitats. In the Ulotrichales he placed the Chaetophoraceae including Pseudendoclonium , the Ulotrichaceae, the

Ulvaceae and six other green algal families.

Fritsch (1935) acknowledged simularities between Ulotrichaceae and the

Chaetophoraceae, but removed the Chaetophoraceae from Ulotrichales and elevated the family to ordinal status. Heterotrichy, the differentiation of the vegetative body into both a prostrate and an erect system of branching filaments, distinguished taxa of the new order, Chaetophorales. Fritsch considered this vegetative differentiation to be taxonomically fundamental and on this basis retained unbranched filamentous forms in the Ulotrichales. He assigned two families, the Chaetophoraceae and the

Trentepohliaceae to the Chaetophorales. In the former were included the “more reduced” forms, or those taxa whose erect systems were diminished or absent. Forms placed in the Trentepohliaceae were the presumably “more primitive” examples that had retained a robust erect system. Fritsch placed Pseudendoclonium in the

Trentepohliaceae presuming a close alliance with Gongrosira.

During the two decades following the compilation by Fritsch, systematics of

filamentous green algae based on general morphology became increasingly confused

and controversial. Smith(1950), Printz, (1964), and Bourrelly, (1966) each proposed

taxonomic systems that differed significantly at the familial and ordinal levels. It was

Bourelly’s system that would be generally accepted and included the more restrictive

orders Ulotrichales, Ulvales and Chaetophorales. Bourelly assigned six families to the

Chaetophorales and divided the Chaetophoraceae into three sub-families. He placed

Pseudendoclonium in the sub-family Leptosiroideae with approximately thirty-five

other genera based on general morphology (Bourelly, 1966).

During the 1970’s, ultrastructural studies of mitosis, cytokinesis and the flagellar

apparatus led to the erection of the class Ulvophyceae to accommodate Ulva and related

genera that shared a number of ultrastructural characteristics. A counter-clockwise

orientation of basal bodies in motile cells and the absence of a phycoplast during

vegetative cell division were the definitive characters of the class (Stewart, Mattox and

Floyd, 1973, Stewart and Mattox, 1978, Mattox and Stewart, 1984, O’Kelly and Floyd,

1984a). A summary of all available ultrastructural information included data from

Pseudendoclonium submarinum, P. basiliense and P. akinetum (O’Kelly and Floyd,

1984b) . These observations supported inclusion of Pseudendoclonium in the Ulotricales

and therefore the Ulvophyceae, but also revealed differences between the three species

that cast doubt on the monophyly of the genus. P. basiliense and P. akinetum possess

minute, diamond-shaped body scales on the surface of zoospores. Such scales are absent

from zoospores of P. submarinum. Also, in P. submarinum and P. basiliense basal

bodies are nearly perpendicular to the long axis of the zoospore while in P. akinetum ,

like Ulothrix zonata, a V-shaped configuration is evident.

The preceeding account cites significant developments in the effort to

reconstruct a natural phylogeny of filamentous green algae relying primarily on light

and electron microscopy. More recently, use of molecular sequence data has both

supported and expanded our hypotheses regarding these phylogenetic concepts. DNA

sequence data from the nuclear-encoded small subunit (SSU) ribosomal RNA gene (18s

rDNA) and others, including genes from the chloroplast, are routinely used along with

morphological and ultrastructural data to infer phylogenetic relationships among the green algae (Friedl 1996, Hayden and Waaland 2002, O’Kelly et al. 2004).

Circumscription of the Ulvophyceae remains controversial as elevation of some orders to class rank has been proposed by several investigators (van den Hoek et al ,

1995). There has also been debate over the inclusion of the Trentepohliales in the

Ulvophyceae (López-Bautista and Chapman, 2003). There is consensus, however, that the Ulvales and Ulotricales form a monophyletic group within the Ulvophyceae, are closely related, and represent early divergent forms of the class ( Zechman et al 1989,

Watanabe et al, 2001). Available data suggest that species of Pseudendoclonium are closely related to other taxa of the Ulotrichales and Ulvales.

Previous phylogenetic studies based on gene sequence data include many genera of the Ulotricales and Ulvales (Friedl, 1996, Hayden and Waaland, 2002, O’Kelly et al

2004a & b, Lindstrom and Hanic, 2005). At the species level, SSU rDNA sequence data are available for Pseudedoclonium basiliense and P. akinetum. Partial SSU rDNA sequence data are available for P. fucicola.

In the current study, SSU rDNA sequence data for the type species of

Pseudendoclonium, P. submarinum are presented. These data are utilized along with previously published data in phylogenetic analyses designed to establish ordinal affiliation of Pseudendoclonium and test monophyly of the genus from a molecular phylogenetic perspective.

CHAPTER 2

MATERIALS AND METHODS

2.1 Isolation and Culture

Specimens of Pseudendoclonium submarinum from Drobak, Norway were

provided by R. Nielsen (Copenhagen) and J. Rueness (Oslo) collected independently in

1999 at Wille’s type locality. Unialgal cultures were developed from this inoculum.

Culture of Pseudendoclonium submarinum began with the preparation of von

Stosch’s enriched seawater medium as previously described (von Stosch 1964).

Salinity of the initial seawater medium at 35 ppt was lethal to all specimens in culture. Many experimental dilutions of seawater media confirmed that optimal growth conditions were realized when salinity was reduced to the natural level found in brackish estuaries. Growth also improved with reduction of the concentrations of added nutrients. The final medium contained seawater diluted by 50% with sterile, distilled water and a 50% reduction of the concentration of added nutrient salts. This modified von Stosch’s enriched seawater medium measured 16 ppt dissolved salts and was utilized exclusively in the algal cultures with the addition of one or more antibiotics.

Culture medium was renewed at least once per month.

Unialgal cultures were grown in 20 X 100 mm glass Petri dishes under fluorescent and incandescent light at an intensity of 5-20 µmol photons · cm -2 · s -1 and a photoperiod of 12 hours darkness / 12 hours light at an average temperature of 16 o C.

Contamination of cultures by unwanted organisms was eliminated by the use of

antibiotics and serial dilution. Diatoms were controlled by addition to the medium of 6

mg/liter germanium dioxide to inhibit frustule development. GeO 2 is not readily soluble in water; the germanium solution was heated to 70 o C with constant stirring to facilitate dissolution. Penicillin or ampicillin and streptomycin were added for control of cyanobacteria and were effective against all but a single strain of a coccoid cyanobacterium. Field collected samples of Pseudendoclonium submarinum were invariably contaminated with a fungus, identified as a species of Cladosporium that thrived in the unialgal cultures of this organism. The anti-fungal amphotericin-B proved to be effective for control of this fungus. This compound is also insoluble in water necessitating pre-dissolution in dimethyl sulfoxide before addition to the medium solution. Elimination of the fungus in cultures with heavy contamination required the use of amphotericin-B at 10 mg/L, or four times the recommended concentration. In new cultures, and cultures from which the fungus had been eliminated, the standard concentration of 2.5 mg/L effectively prevented fungal growth. The concentrations of nutrients, vitamins and antibiotics are given (Table 2.1).

2.2 Microscopy

Cultures were monitored using a Nikon SMZ800 dissection microscope equipped with an Excel Technologies F0-150 illumination system (Excel Technologies,

Enfield, CT, USA). Cellular morphology was observed using an Olympus Model BH-2 compound microscope. Digital images from both microscopes were captured utilizing a

Model 18.2 Color Mosaic digital camera (Diagnostic Instruments Inc., Sterling Heights,

MI, USA).

2.3 DNA Extraction

200 mg samples were scraped from Petri dishes of pure algal cultures using a stainless steel razor blade after draining of the liquid medium and partial air drying.

Material of the species from several culture dishes were typically harvested in order to obtain sufficient material for DNA extraction. Samples were air-dried for several hours to a weight of approximately 75 mg. The moisture content of the harvested material proved to be a critical factor in the disruption process and extensive air-drying of the material was required. The samples were next placed in a 2 ml microcentrifuge tube containing 8-10 2.3 mm glass beads and frozen in liquid nitrogen for 30 seconds. Cells were disrupted using a Mini Bead Beater (Biospec Products, Bartlesville, OK, USA) at maximum speed (46,000 Hz) for one or two 20-second cycles with an additional cooling step between cycles of 30 seconds in liquid nitrogen. After bead mill processing, the material was again frozen in liquid nitrogen for 30 seconds at which time lysis buffer was immediately added. Genomic DNA was then extracted and purified using the DNeasy Mini Plant Kit (Qiagen Inc., Valencia, CA, USA) or the

Phytopure DNA Purification Kit (Tepnel Life Sciences, Manchester, UK) according to the manufacturer’s directions. The Phytopure kit consistently yielded genomic fragments of greater length than did the Qiagen kit and the use of template DNA from the former kit increased PCR efficiency significantly.

2.4 DNA Amplification

The nuclear encoded small-subunit ribosomal DNA (18S rDNA) gene was amplified using two separate polymerase chain reaction series that generated fragments of approximately 800 and 1000 base pairs in length. This strategy was utilized after

realization that PCR product from the smaller fragments had a much higher DNA concentration and purity than product obtained by amplifying the gene as a single fragment.

A 200 µL polymerase chain reaction master mix was prepared containing 129

µL sterile distilled water, 20 µL Amplitaq PCR BufferII (Applied Biosystems, Foster

City, CA, USA), 16 µL 25mM MgCl 2, 4 µL of each 0.2mM dNTP (New England

Biolabs Inc., Ipswich, MA, USA), and 8 µL of each oligonucleotide primer (Integrated

DNA Technologies Inc., Coralville, IA, USA) at a concentration of 10 µM. Immediately prior to each reaction 1.0 units of AmpliTaq Gold DNA Polymerase (Applied

Biosystems) and 2 µL of genomic DNA were added and the mix divided into four 50

µL reactions in 0.5 ml reaction tubes. Reactions were run in a Mini Cycler (MJ

Research Inc., Watertown, MA, USA) with a reaction profile of 3 min initial denaturing and enzyme activation at 95 o C, and 35 cycles of 45 seconds denaturing at 94 o C, 45

seconds annealing at 50 o C and 90 seconds extension at 72 oC with a final extension of 5 min. at 72 o C. Four 50 µl reactions were subsequently pooled for analysis.

2.4.1 PCR Primers

The two regions of the SSU gene that were separately amplified were designated

18sA and 18sB. The 18sA fragment includes positions 2 – 811 and was amplified with a forward primer from a previously published study (O’Kelly et al 2004). A compatible reverse primer was chosen from conserved regions of the previously published sequence of Pseudendoclonium basiliense (Genbank accession Z47996) with the aid of the IDT website tools (www.idt.com). 18sB includes positions 788 – 1771 and in this

case both primers were designed for this study. A complete list of primers used in this study is given (Table 2.2).

2.5 Detection and Analysis

PCR products were visualized by gel electrophoresis in 1%, 50 ml agarose gels stained with 1.5 µg ethidium bromide solution (Sigma, St. Louis, MO) and examined in

ultraviolet light. 3 µL of PCR product were typically run for 90 min. at 100 volts DC.

DNA concentrations were estimated by comparison of band intensity with known

concentrations present in a 1kb DNA ladder (New England BioLabs, Ipswich, MA).

2.5.1 Purification

PCR products were first prepared for sequencing using the QIAquick PCR

Purification Kit (Qiagen) but products purified in this manner resulted in ambiguous

and unreliable sequence data. Subsequent purification utilizing the QIAquick Gel

Extraction Kit (Qiagen) yielded PCR product of sufficient purity to generate reliable

sequence. For the gel extraction protocol, 35-50 µL of PCR product were loaded into

each of two adjacent lanes of a 100 ml agarose gel along with 10 µL of loading dye.

After electrophoresis, the resulting bands were excised from the gel and purified

according to the manufacturer’s instructions.

2.5.2 Sequencing

Purified products were sequenced utilizing the dideoxy chain termination method (Sanger et al. 1977) in an ABI automated sequencer at the University of Maine,

Orono sequencing facility. Sequencing was performed primarily in the forward direction utilizing sequencing primers spaced approximately every 400 bp over the

length of the gene. Reverse sequencing primers at approximately 300 bp from the beginning of each fragment and overlap of the forward sequences provided verification.

2.5.3 Sequencing Primers

Sequencing primers were chosen from previously published work or designed for this study in the above fashion as noted (Table 2.2). PCR primers seldom yielded acceptable results in the sequencing reactions. Sequencing primers that included the latter end of the PCR primer sequence and extended beyond it, however, returned excellent results in most cases.

2.5.4 Sequence Editing

Editing was effected by alignment of the raw chromatogram data with previously published sequences of the closely related species Ulothrix zonata and

Pseudendoclonium basiliense in Clustal X (Higgins and Sharp 1988; Thompson et al.

1997; Thompson et al. 1994). Each site of the newly generated sequence data was then evaluated with respect to the integrity of the chromatogram signal, its alignment with the known sequence data and its alignment with overlapping data. Editing was performed in BioEdit (Hall 1999). Each edited data file was sequentially added to a master alignment in Clustal X until the complete sequence of the two regions was realized.

2.6 Phylogenetic Analysis

Molecular phylogenetic analyses presented in this study are modelled, to a large extent, after that of a previously published study of the Ulvophyceae (Hayden and

Waaland 2002). Hayden and Waaland investigated the systematics of the orders Ulvales and Ulotrichales using partial SSU rDNA sequence data and sequence data from the large subunit of the chloroplast encoded RUBISCO gene. Phylogenetic analyses were presented for twenty taxa first using separate sequence data from each of the genes and a third analysis using a combined data matrix from both genes. Tree topologies were nearly identical in all analyses and clearly recovered the two orders as well-supported monophyletic groups. Data from Pseudendoclonium fucicola included in their study clearly placed this species within the Ulvales clade. This brought into question the monophyly of the genus since a previous molecular phylogenetic investigation had shown a close affiliation of Pseudendoclonium basiliense with the Ulotricales (Friedl

1996) .

The suggestion of Hayden and Waaland that Pseudendoclonium is polyphyletic is tested in the current study by implementation of two analyses similar in scope to theirs, but also including data from P. submarinum, P. basiliense and P. akinetum.

Importantly, and a primary objective of this study, these analyses will infer an ordinal alliance of the type species of Pseudendoclonium (P. submarinum ) which is unresolved.

Resolution of the phylogenetic position of the type species must preclude any discussion of the systematics of the genus.

The partial 18s sequences generated by Hayden and Waaland represent a 753 bp fragment spanning the variable 4 region of the gene and represent only 42 percent of the

gene. Since this partial sequence data is the only data available for P. fucicola and the closely related genera Tellamia and Kornmannia , two separate analyses of 18s rDNA

sequence data are presented in this study. For most genera in the Hayden and Waaland

analysis, full 18s sequence data is available for species other than those for which these

authors generated partial sequences. This allows presentation in this study of two

similar analyses, one using only partial sequence data including that of P. fucicola, and

another utilizing complete SSU sequence data that excludes this species, Kornmannia

leptoderma and Tellamia contorta .

Sequences were aligned in Clustal X for both analyses and edited by eye using

BioEdit. Sequences were truncated to equal lengths. Gap opening and gap extension

penalties were set to the default values. Alignment files were exported in the NEXUS

format for use by PAUP*.

Heuristic searches were first conducted under the MP criterion in PAUP* for

both analyses with tree bisection-reconnection and MULTREES options in effect.

Character state changes and nucleotide positions were unweighted and introduced gaps

were coded as missing data. Ten replicate searches with randomized taxon input were

conducted using the random stepwise-addition method. Branch support was estimated

by nonparametric bootstrap analysis (Felsenstein 1985) with 2000 replicates.

Appropriate evolutionary models for the maximum likelihood analyses were

determined by hierarchical likelihood ratio tests of each data matrix using Modeltest

v.3.7 (Posada and Crandall 1998). From the full-length sequence data set the chosen

model was a Tamura-Nei 1993 model with equal base frequencies, an estimated

proportion of invariable sites (I = 0.5815), and gamma distributed rate variation among

sites ( α = 0.6095). The substitution rate matrix is as follows: A-C = 1.0000, A-G =

2.5585, A-T = 1.0000, C-G = 1.0000, C-T = 4.3987, G-T = 1.0000.

In the Modeltest analysis of the partial sequence data set, the chosen model was

the TIMef + I + G model known as the Transversion model with equal base frequencies,

an estimated proportion of invariable sites (I = 0.6337), and gamma distributed rate

variation among sites ( α = 0.5914). The substitution rate matrix is: A-C = 1.0000, A-G

= 3.7630, A-T = 2.2750, C-G = 2.2750, C-T = 8.5392, G-T = 1.0000. Heuristic searches

were conducted with the same options in effect as in the MP searches. Bootstrap

analysis with 100 replications under the ML criterion was used to estimate branch

support (Felsenstein 1985).

CHAPTER 3

RESULTS

3.1 General

The complete nucleotide sequence of the 18s SSU rDNA gene for

Pseudendoclonium submarinum Wille from Drobak, Norway is identified (Table 3.1).

Sequence data has been deposited in Genbank accession number EF591129.

Alignment of the ingroup taxa for both the partial and full sequence analyses was straightforward; few gaps were introduced. The Trebouxiophytes, Chlorella vulgaris and Myrmecia astigmatica were included as outgroup taxa following Hayden and Waaland (2002). Sequences for these taxa introduced the shortest branches of all available outgroups from the Ulvophyceae. Insertions in the outgroup taxa were removed at several sites to prevent resulting gaps in the entire alignment.

Maximum likelihood trees inferred from both partial and full-length SSU data recovered the orders Ulvales and Ulotricales as monophyletic sister groups. This assessment must be qualified by the following two facts. The first is that bootstrap support for the branch leading to the Ulotricales in both analyses under the ML criterion is weak. Secondly, O’Kelly et al. (2004a) show that when certain coccoid ulotricalean

genera (not included here) are included in an analysis spanning both of these orders,

Ulvales are recovered as a monophyletic group within a paraphyletic order Ulotricales.

Support for monophyly of the Ulvales was robust in these analyses. Relationships

among taxa closely allied with Ulothrix zonata are not resolved in either analysis due to

the high degree of conservation in the 18s gene. Among these taxa, putative species

may be separated by less than 10 substitutions in the entire gene.

3.2 Partial-Sequence Analysis

Pseudendoclonium fucicola, Tellamia contorta and Kornmannia leptoderma are excluded from the full-length SSU sequence analysis. Only partial SSU sequence data are available for these taxa. They are included in an analysis of partial SSU sequence data, along with partial-sequence data from all taxa included in the full-length sequence analysis. It has previously been shown that P. fucicola, T. contorta and K. leptoderma group together with Blidingia to form a distinct clade within the Ulvales (Hayden and

Waaland 2002). This clade is recovered as sister to the clade containing the Ulvaceae and Acrochaete. These clades represent separate lineages within the Ulvales. The partial sequence analysis indicates that P. submarinum groups within the clade containing P. fucicola, T. contorta, K. leptoderma, P. salina and Blidingia dawsonii. Inclusion of taxa known to group closely with P. fucicola is useful in the evaluation of the evolutionary relationship between P. submarinum and P. fucicola.

Alignment of the partial-sequence data consisted of 749 total characters of which 611 were constant, 138 were variable and 102 were parsimony-informative.

Heuristic searches under the MP criterion returned 32 most parsimonious trees of 281 steps in length. Three trees shared topology with the maximum-likelihood tree (Fig.

3.1). The most significant difference relative to this discussion between the MP and ML trees is the placement of Pirula salina with respect to Pseudendoclonium submarinum.

In one half of the MP trees, P. salina is basal with regard to P. submarinum, while in the other sixteen MP trees these species are placed sister to each other as they are in the

ML tree. The remaining two instances of noncongruity among MP trees involve relationship between Percursaria and Ulvaria and that between Acrosiphonia and

Gloeotilopsis . These inconsistencies do not directly impact the present discussion of

Pseudendoclonium.

The likelihood score of the ML tree from partial-sequence data is substantially lower (-ln L = 2541.69159) than that of the full-sequence tree (-ln L = 5817.70501) and reflects the lack of phylogenetic signal in the partial sequence data. The high degree of conservation within the SSU gene among taxa that group closely with Ulothrix precludes resolution of the generic relationships among Pseudendoclonium basiliense,

P. akinetum, Trichosarcina, and Ulothrix . The data support only the conclusion that P. basiliense and P. akinetum group within the ulotricalean lineage and are closely related to Ulothrix zonata and Trichosarcina mucosa .

On the other hand, the ulvalean clade containing the type species, P. submarinum and P. fucicola is well supported under both the MP and ML criteria.

Evolutionary relationships among Tellamia, Blidingia, Kornmania, Pirula and the two species of Pseudendoclonium are well resolved. P. submarinum and Pirula salina form a subclade that is sister to the remainder of the group, though these branches are weakly supported in the bootstrap analyses. Kornmania and Blidingia occupy positions basal to

a terminal subclade formed by P. fucicola and Tellamia contorta. Bootstrap support for

the latter two branches is robust.

Although the sequences of P. submarinum and P. fucicola are recovered as part

of this clade, their phylogenetic positions within in the clade are distant. Comparison of

the 749 bp partial SSU sequence data of these two species reveals that their sequences

differ by 36 base pairs. The difference would be significantly greater if all variable

regions of the P. fucicola gene were considered, and greater than distances that typically

separate genera within these two orders.

3.3 Full-Sequence Analysis

Alignment of the full sequence SSU data for the 22 taxa included in this study

was also straightforward. There were 1683 total characters of which 1344 were

constant, 339 were variable and 232 were parsimony-informative. Heuristic searches

under the MP criterion returned 2 most parsimonious trees of 642 steps in length. One

of these trees shared topology within the Ulotricales and the ulvalean clade containing

P. submarinum with the maximum-likelihood tree shown (Fig. 3.2). This tree differed

from the ML tree in placement of Enteromorpha, Ulva, Percursaria and Ulvaria.

Relationships within the Ulvaceae are not well resolved in this analysis. However

bootstrap support for monophyly of the Ulvales and for the Ulvaceae is robust.

The full-sequence analysis recovers P. submarinum and therefore the genus as

part of the Ulvalean lineage. P. submarinum and Pirula salina form a terminal subclade

in a monophyletic group that includes Blidingia and Bolbocoeleon. Blidingia dawsonii

occupies a position sister to the P. submarinum and P. salina clade and bootstrap

support for the branch leading to these three taxa is robust. Tree topology differs

between the partial and full sequence analyses with respect to the position of

Bolbocoeleon piliferum . In the partial sequence analysis this species occupied a position

basal to the entire clade that includes P. submarinum . Here, Bolbocoeleon is placed in a

position sister to the remainder of the clade and boostrap support for the entire clade is

poor. Support for the clade excluding Bolbocoeleon in the partial sequence tree was

robust. Placement of Bolbocoeleon within this clade seems equivocal. Excepting the

enigmatic position of Bolbocoeleon , relationships within the clade are well resolved.

The Ulotrichales were again recovered as a monophyletic group in the full-

sequence analysis. Bootstrap support for the ulotrichalean clade was robust under the

MP criterion but poor in the ML analysis. Two subclades containing Acrosiphonia and

Gloeotilopsis received robust bootstrap support under both criteria. Generic relationships among Pseudendoclonium basiliense, P. akinetum , Monostroma grevillei,

Trichosarcina mucosa and Ulothrix zonata were not clearly resolved due to low

sequence variation among these taxa. The polytomy including P. basiliense, U. zonata,

M. grevillei, and T. mucosa recovered in the partial sequence analysis is better resolved utilizing the full sequences. However, bootstrap support for the topology of the tree in this region is poor. The sequence data support a close affinity of P. basiliense and P. akinetum with Ulothrix and therefore their inclusion within the Ulotricales.

CHAPTER 4

DISCUSSION

4.1 Generic

The objectives of this study were: (1) determination of the taxonomic position of

Pseudendoclonium inferred from analysis of SSU rDNA sequence data obtained from isolates of P. submarinum, the type species and (2) molecular phylogenetic evaluation of the monophyly of the genus with respect to three species currently assigned to

Pseudendoclonium for which SSU rDNA sequence data are available.

Phylogenies reconstructed in the present analyses clearly support the hypothesis that Pseudendoclonium, as it is currently circumscribed, is polyphyletic. These analyses are based on SSU rDNA sequences that include data from the type species of the genus.

These data clearly indicate that the genus requires redefinition. The three species currently assigned to Pseudendoclonium discussed in this study must be removed from the genus. P. submarinum and P. fucicola are part of the ulvalean lineage. Based on the molecular sequence data generated and reviewed during this study, these two species are not congeneric. From a genetic perspective, P. basiliense and P. akinetum are clearly ulotrichalean, and group closely with Ulothrix zonata despite significant morphological differences. On the basis of molecular analysis, P. basiliense and P. akinetum should be placed in a single genus as they are certainly closely related.

Unquestionably, P. basiliense and P. akinetum lack affinity with Pseudendoclonium and require an alternate generic identity.

Pseudendoclonium submarinum, P. fucicola, P. basiliense and P. akinetum are remarkably similar in vegetative and reproductive morphology. All are minute forms

with cells 5 to 10 µm in length and filaments of only 5 to 20 cells in length. They share

a heterotrichous growth habit having irregularly branching vertical filaments arising

from a prostrate basal disc of cells some of which are rhizoid-like in appearance and

function. All possess a single, parietal chloroplast containing one distinctive pyrenoid.

Species have been separated based on general morphological and reproductive

characteristics. These characteristics include overall size, proportion of basal and

vertical filaments, extent of branching and akinete characteristics. Morphological

characteristics such as these may be subject to environmental variability; their

usefulness as taxonomic markers is questionable.

P. submarinum and P. fucicola are primarily marine species, though P.

submarinum occurs in brackish estuaries. P. basiliense and P. akinetum are freshwater

epiphytes of a number of aquatic angiosperms (Vischer 1926, Tupa 1974).

4.2 Specific

Pseudendoclonium remained monospecific until Vischer (1926) described P.

basiliense. Vischer isolated the alga from a small pool in a garden stream at Bâle

(Basel) Switzerland. Vischer’s cultures have been lost, however Tupa (1974) described

an epiphytic alga from various freshwater locations in Texas that she believed to be P.

basiliense . Details of vegetative and reproductive morphology of P. basiliense are

nearly identical to those of P. submarinum and P. fucicola . Mature, erect filaments in all

three species become biseriate or pleurococcoid in appearance. This is a distinguishing

feature that Wille noted in P. submarinum. Filaments of P. basiliense , both prostrate

and erect, are several cells longer than those of P. submarinum and P. fucicola so that

the thallus is generally larger (Tupa 1974).

Zoospore germination is bipolar in P. basiliense in contrast to the unipolar germination observed in P. submarinum. Zoospores of P. basiliense are

quadriflagellate; no biflagellate motile cells have been described (Tupa 1974).

Ultrastructural studies reveal that zoospores of P. basiliense are covered with distinct

body scales, a ulotricalean characteristic absent from zoospores of P. submarinum

(Mattox and Stewart 1973, Floyd and O’Kelly 1984).

Tupa (1974) also collected and described two new species which she assigned to

Pseudendoclonium . P. akinetum and P. prostratum are freshwater epiphytes collected

from a number of aquatic hosts in widely separated localities in Texas and Illinois. P.

prostratum is so named because of the virtual absence of upright filaments. Its thallus is

described as “a richly branched attached prostrate filament, a single layered, pseudo-

parenchymatous disc with or without erect branches” (Tupa 1974). Lack of sequence

data from this species precludes any further discussion in the present study.

Pseudendoclonium akinetum (Tupa 1974) is distinguished from P. submarinum,

P. fucicola and P. basiliense by its production of characteristic akinetes, and the

spherical shape of its cells. It is further distinguished by the paucity of erect filaments,

the shorter length of filaments and the lack of biseriate or “pleurococcus-like” mature

filaments. Akinetes are thick-walled and often surrounded with a granular, flaky, dark

reddish-brown material. These akinetes measured up to 40 µm diameter and are much

larger than any described for the other species (Tupa 1974, Yarish 1975). Like

P.basiliense, the zoospores of P. akinetum possess tiny, organic body scales (Floyd and

O’Kelly 1984).

SSU rDNA sequence data support the hypothesis that P. basiliense and P.

akinetum are closely related, but distinct species. These sequences differ by seven

substitutions of 1683 positions considered. This difference is comparable to

interspecific variation within other genera of the Ulotricales for which sequence data are

available. However, the SSU gene is highly conserved within the ulotricalean lineage.

The single gene is not sufficiently variable to reliably recover phylogeny at the generic

and specific levels in all instances. SSU rDNA sequences of P. akinetum and Ulothrix

zonata, for example, differ at only 9 positions, a difference that corresponds to a

specific difference within several other putative genera of the lineage. Sequence data

support only the conclusion that P. basiliense, P. akinetum and U. zonata are very closely related organisms within the taxonomic concept of the Ulotrichales. Thus the contention that P. basiliense and P. akinetum be considered congeneric taxa relies heavily on vegetative and reproductive morphological evidence.

Ulvella fucicola was described by Rosenvinge (1893), a marine epiphyte of

Fucus inflatus (= F. evanescens ) collected from the west coast of Greenland at

Egesdesminde. Wille (1909) transferred U. fucicola and a second species, U. confluens , to Pseudopringsheimia separating both from Ulvella and Pseudulvella based on “the presence (in Pseudopringsheimia ) of rhizoidal outgrowths from the base penetrating the host” (Setchell and Gardner 1920). It is noteworthy that Wille did not consider transferring U. fucicola to Pseudendoclonium at this time since he had described P. submarinum nine years previously. Nielsen (1980) examined several isolates of

Pseudopringsheimia fucicola in culture for comparison of this alga with P. submarinum . She distinguished the two species by the position and shape of the

sporangia. Sporangia of P. fucicola are described as being conical in shape and

developing only from the uppermost cells of the vertical filaments. Several authors who

previously studied P. submarinum including Wille, noted that its sporangia develop

from any vegetative cell of the upright filaments. Citing numerous morphological

similarieties between the two species, Nielsen transferred Pseudopringsheimia fucicola

to Pseudendoclonium but made little note of species distinction.

Nielsen was the first author to observe both quadriflagellate and biflagellate

motile cells in both organisms. In one isolate of P. submarinum she observed that

biflagellate swarmers were of two different sizes and were released from larger

sporangia than were the quadriflagellate zoospores. Nielsen did not determine whether

biflagellate and quadriflagellate swarmers arose from separate thalli. Biflagellate motile

cells observed in cultures of P. fucicola were of equal size and were released from

separate, morphologically similar thalli. This observation supports isomorphic

alternation. However, copulation between biflagellate swarmers was not observed for

either species.

Comparison of SSU rDNA sequence data from Pseudendoclonium submarinum

and P. fucicola reveal a level of sequence variation that is comparable to or greater than

variation between putative genera of this lineage. Based on these data, P. submarinum

and P. fucicola cannot be considered congeneric. P. fucicola must therefore be

removed from Pseudendoclonium.

The principal morphological features that distinguish the Ulotricales and

Ulvales are present only in sexually reproductive forms. A Codiolum-type zygotic stage

in the life cycle typifies the life history of members of the Ulotricales. This club-shaped

zygote contains the only diploid cell of the life history of these species. In contrast, sexually reproductive algae of the Ulvales, where known exhibit an isomorphic, diplohaplontic life cycle in which haploid gametophytes alternate with morphologically identical diploid sporophytes. However, sexual reproduction has not been described for any of the four taxa considered in this study and considerable knowledge of life histories is lacking. Morphological features that might be used to support the molecular data regarding ordinal affiliation of these taxa are therefore few. Following is a summary of these characters.

The occurrence of minute, organic body scales on the surface of zoospores is one feature that supports ordinal separation of Pseudendoclonium basiliense and P. akinetum from P. submarinum. These scales are very similar to the inner body scales of the Prasinophyceae, a green algal class of unicellular organisms considered ancestral with respect to the Ulvophyceae. The scales are thus considered a vestigal character

(van den Hoek et al. 1995). The scales are present on zoospores of P. basiliense, P.

akinetum and Ulothrix zonata. They are not present on zoospores of P. submarinum and

have not been reported to occur on those of P. fucicola or any other algae of ulvalean

lineage. The presence of the scales supports the close relationship of P. basiliense, P. akinetum and Ulothrix indicated by the molecular data. The absence of scales from P. submarinum , and P. fucicola separates these taxa from P. basiliense and P. akinetum.

This is congruent with the view that the Ulvales are the more derived lineage of the two

orders.

Both biflagellate and quadriflagellate zoospores have been observed in cultures of P. submarinum and P. fucicola , but only quadriflagellate swarmers have been noted in P. basiliense and P. akinetum (Vischer 1926, Tupa 1974, Neilsen 1980).

Zoospore germination is unipolar in P. submarinum and P. fucicola while

bipolar germination has been described for both P. basiliense and P. akinetum (Vischer

1933, Tupa 1974, Neilsen 1980).

Two morphological differences support generic separation of Pseudendoclonium

submarinum and P. fucicola. The zoosporangia of P. fucicola develop only in the most

distal cells of the upright filaments (Nielsen 1980). P. fucicola shares this feature with

species of Pseudopringsheimia, from which it was transferred by Nielsen (1980).

Zoosporangial development in P. submarinum, on the other hand, may occur in any cell

of the mature, upright filaments. P. submarinum is further distinguished from P.

fucicola by the size of biflagellate motile cells. Both organisms produce biflagellate and

quadri-flagellate motile cells. The biflagellate cells of P. submarinum are of two distinct

sizes while those of P. fucicola are of equal size (Nielsen 1980).

CHAPTER 5

CONCLUSION

5.1 Evolutionary Considerations

Molecular phylogenies recovered in this study are inconsistent with long-

standing and widely-accepted taxonomy regarding the circumscription of

Pseudendoclonium . These results were unexpected. Phylogenies recovered using molecular sequence data are typically consistent with traditional phylogenies inferred from morphological diversity. This is evident in previous molecular phylogenetic investigations of the Ulotrichales and Ulvales (O’Kelly et al 2004a, Friedl 1996,

Zechman et al 1990). Gloeotilopsis, Achrocheate, Phaeophila and Acrosiphonia are examples of genera that were first circumscribed on the basis of morphological criteria.

Subsequent molecular-based phylogenies clearly recover these genera as monophyletic groups. Morphological similarity among the “species” of Pseudendoclonium discussed in this study is sufficient to warrant their inclusion in a single genus. Here, molecular evidence does not support morphological phylogeny. Possible explanations for this dichotomy are difficult to test experimentally and must remain, at least presently, somewhat speculative.

The ulotrichalean and ulvalean lineages are widely considered to be early- diverging lineages among the green algae. Fritsch (1935) hypothesized that the unbranched filamentous form of Ulothrix represents a single evolutionary step beyond the motile, unicellular condition characteristic of the ancestral Prasinophyceae. The heterotrichous condition, characteristic of the organisms discussed in this study, is a simple elaboration of a subsequent innovation in the evolution of green algae; the

branched filament. These ancient organisms or their immediate ancestors were among the first eukaryotic, multicellular autotrophs. It is likely that these rudimentary green algae predate the appearance of vascular plants by several hundred million years.

The heterotrichous growth habit is a familiar and evolutionarily convergent form evident in several major lineages of algae. The simplicity and efficiency of a prostrate system of filaments for substratum attachment and vertical filaments extending toward insolation has evolved many times. Considering the antiquity of the ulotrichalean and ulvalean lineages, there has been ample evolutionary time for any number of organisms to converge upon this common form, persist or become extinct and for others to subsequently evolve as well. This has likely occurred several times and separately within both the ulotrichalean and ulvalean lineages. The significant number of synapomorphies in cytological structure among the organisms considered in this investigation support this view.

5.2 Specific Assignment

Pseudendoclonium is typified in this study by elucidation of the SSU rDNA

gene sequence for Pseudendoclonium submarinum Wille. Unialgal cultures of this

organism were established with innoculum collected at the type locality in Drobak,

Norway. Genomic DNA extracted from the unialgal cultures was used to generate

sequence data. Phylogenetic analyses place P. submarinum within the Ulvales as part of

a clade representing a lineage within the Ulvales that is separate from that containing

the Ulvaceae. Based on the sequence data, Pseudendoclonium shares a close

relationship with Pirula, Kornmannia and Blidingia within this clade.

Based on partial SSU sequence data, Pseudendoclonium fucicola groups within the same Ulvalean clade as P. submarinum but is separated from the type species by several other genera in the phylogenetic analysis. Therefore, P. fucicola cannot be retained in Pseudendoclonium and requires generic reassignment. P. fucicola forms a terminal subclade in the partial-sequence tree with Tellamia contorta. A BLAST search of the nucleotide database in GenBank confirms that T. contorta is the most closely related alga to P. fucicola among organisms for which sequence data are available.

However, the two sequences differ at 14 of the 753 sites in the partial sequences. The distance would likely be considered generic were the full SSU sequences evaluated.

Therefore, no generic assignment for Pseudendoclonium fucicola is suggested here.

The freshwater ulotrichalean species currently known as Pseudendoclonium basiliense and P. akinetum are separated from P. submarinum at the ordinal level and therefore require generic reassignment. P. basiliense and P. akinetum share a high degree of SSU sequence similarity with Hazenia mirabilis and Trichosarcina mucosa.

SSU sequences of these four species are identical at all but 14 of 1683 sites. As previously noted, the SSU rDNA gene is highly conserved among “core” ulotrichalean genera and is not sufficiently variable to resolve relationship at the generic level in many instances. The sequence of P. basiliense for example, differs from that of P. akinetum at 7 sites but differs from H. miribilis at only six. Resolution of generic relationships among these taxa requires further genetic analysis with more variable genes.

From a morphological perspective, Pseudendoclonium basiliense and P. akinetum share higher similarity with Trichosarcina mucosa than either does with

Hazenia miribilis. Hazenia is a soil alga with branched filaments enclosed in a tube-like sheath of mucilage with terminal conical cells. Sexual reproduction occurs via isogamous biflagellate gametes and zoospores are unknown. No ultrastructual data is available for H. mirabilis.

Trichosarcina has been considered by several authors to be a close relative of

Pseudendoclonium basiliense. Trichosarcina differs from P. basiliense in its production of a single zoospore per cell, a lack of heterotrichy and its multiseriate condition. Like

P. basiliense and P. akinetum, reproduction occurs via quadriflagellate zoospores and sexual reproduction is unknown. In a comparative study of cell division, Mattox and

Stewart (1974) found that the details of mitosis and cytokinesis are so alike in T. mucosa and P. basiliense that “a single text shall suffice for both.” Based on genetic, ultrastructural and morphological simularities, P. basiliense and P. akinetum are tentatively refferred to Trichosarcina as Trichosarcina basiliense and Trichosarcina akinetum.

Collins (1909) cited the first record of Pseudendoclonium submarinum in North

America from the coasts of three New England states. This alga was collected during

2005 for purposes of this study from Crane’s Salt Marsh in Ipswich, Massachusetts.

Unialgal cultures of this North American organism were morphologically indistinguishable from the culture material established with innoculum from Drobak,

Norway. Analysis of SSU rDNA sequence data, however, does not support Collins’ suggestion that the North American alga he identified is P. submarinum. Additionally,

the sequence data do not support assignment of this alga to Pseudendoclonium.

Moreover, these data suggest that the North American alga currently recognized as P.

submarinum is distinct, at the generic level, from P. fucicola, P. basiliense, P. akinetum and P. submarinum.

Resolution of the identity of the North American alga, repeatedly cited as

Pseudendoclonium submarinum Wille from Norway, represented in this study by

material from Ipswich, Massachusetts, requires taxonomic separation from the Wille

alga. The alga appears Ulvalean and separated from Pseudendoclonium submarinum

and Pseudendoclonium fucicola on the basis of SSU rDNA sequence data.

Table 1.1 The genus Pseudendoclonium. ______

1 Pseudendoclonium akinetum Tupa 2 Pseudendoclonium basiliense Vischer 3 Pseudendoclonium dynamenae Nielsen 4 Pseudendoclonium fucicola (Rosenvinge) R. Nielsen 5 Pseudendoclonium informe P. Dangeard 6 Pseudendoclonium laxum John & Johnson 7 Pseudendoclonium murale Brand 8 Pseudendoclonium prostratum Tupa 9 Pseudendoclonium submarinum Wille

Source: Index Nominum Algarum ______

Table 2.1 Composition of modified von Stosch’s Enriched Seawater Medium ______SALTS CONCENTRATION / LITER

NaNO 3 21.25 mg Na 2HPO 4 · 12H 2O 5.375 mg FeSO 4 · 7H 2O 139.0 µg MnCl 2 · 4H 2O 9.90 µg Na 2EDTA · 2H 2O 1.86 mg Vitamins Thiamine-HCl 0.20 mg Biotin 1.00 µg B12 1.00 µg Antibiotics Penicillin or Ampicillin 100,000 units Streptomycin 100.0 mg GeO 2 6.0 mg Amphotericin B 2.5 mg

Table 2.2 Oligonucleotide primers used in this study.

Region Primer Direction Annealing Sequence Reference position 5’ 3’

PCR primers

SSU Pseud A1 F 2 – 19 CTGGTTGATTCTGCCAGT b R 813 R 787 – 811 AGTCCTGTCGTGTTATCCCATGCT a F 790 F 788 – 813 AGCATGGGATAACACGACAGGACT a R 1773 R 1747 – 1771 CAGGTTCACCTACGGAAACCTTGT a

Sequencing primers

SSU NS 1 F 18 – 36 GTAGTCATATGCTTGTCTC c 18s 1b F 359 – 379 GGAGAATTAGGGTTCGATTCC a Rseq 295 R 276 – 299 AGTTGAATGAAACATCGCCGGCAC a Seq 3.6 F 825 – 848 TCTGGCCTATCGTGTTGGTCTGTA a Seq 4 F 1276 – 1298 TGAATGGCCGTTCTTAGTTGGT a NS7m F 1422 – 1438 GGCAATAACAGGTCTGT c Rseq 1035 R 1024 – 1047 TCCCTAGTCGGCATCGTTTATGGT a

References: a: primer designed for this study; b: O’Kelly et al 2004; c: Friedl 1996; Numbering based on the 18s SSU sequence of Ulothrix zonata, GenBank accession number Z47999 ______

Table 3.1 SSU rDNA sequence of Pseudendoclonium submarinum. ______

ORIGIN

1 tctggttgat tctgccagta gtcatatgct tgtcttaaag attaagccat gcatgtctaa 61 gtataaactg cttatacggc gaaactgcga atggctcatt aaatcagtta gagtttattt 121 gatggtacct tactactcgg ataaccgtag taaagctaca gctaatacgt gcgcagatcc 181 cgactcacga agggacgtat ttattagatt caagaccgac cgtgcttgca cgctcttggt 241 gaatcatggt aacttcacga atcgcacggc ctcgtgccgg cgatgtttca ttcaactttc 301 tgccctatca actttcgacg gtagtataga ggactaccgt ggtagtaacg ggtgacggag 361 aattagggtt cgattccgga gagggagcct gagaaacggc taccacatcc aaggaaggca 421 gcaggcgcgc aaattaccca atcctgacac agggaggtag tgacaaaaaa tatcaatact 481 gggcctcatg gtccggtaat tggaatgagt acaatctaaa tccgttaacg aggatccatt 541 ggagggcaag tctggtgcca gcagccgcgg taattccagc tccaatagcg tatatttaag 601 ttgttgcagt taaaaagctc gtagttggat ttcgggtggg ctgcgccggt ctcctaacgg 661 ttgtactggc gacgcctgcc ttgctgccgg ggacgggctc ctgccttaac tgtcgggacc 721 cggaatcggc gacgttactt tgagtaaatt agagtgttca aagcaagcct acgctctgaa 781 tataatagca tgggataaca cgacaggact ctggcctatc gtgttggtct gtaggaccgg 841 agtaatgatt aagagggaca gtcgggggca ttcgtattcc gttgtcagag gtgaaattct 901 tggatttacg gaagacgaac atctgcgaaa gcatttgcca aggatgtttt cattgatcaa 961 gaacgaaagt tgggggctcg aagacgatta gataccgtcg tagtctcaac cataaacgat 1021 gccgactagg gattggcgga agttcttttg atgactccgc cagcacctca tgagaaatca 1081 aagtttttgg gttccggggg gagtatggtc gcaaggctga aacttaaagg aattgacgga 1141 agggcaccac caggcgtgga gcctgcggct taatttgact caacacggga aaacttacca 1201 ggtccagaca tgcgaaggat tgacagattg aaagctcttt cttgattgta tgggtggtgg 1261 tgcatggccg ttcttagttg gtgggttgcc ttgtcaggtt gattccggta acgaacgaga 1321 cctcagcctg ctaaataggt tcgtctgctc cggcagtcga ctatcttctt agagggactg 1381 ttggcgtcta gccaatggaa gtatgaggca ataacaggtc tgtgatgccc ttagatgttc 1441 tgggccgcac gcgcgctaca ctgacacgtt caacaagttc cttgtccgaa aggtctgggt 1501 aatctttgaa accgtgtcgt gatggggata gaacattgca attattgttc ttcaacgagg 1561 aatgcctagt aagcgcgagt catcatctcg cgttgattac gtccctgccc tttgtacaca 1621 ccgcccgtcg ctcctaccga ttgaacgtgc tggtgaagcg ttaggactgg actgttggct 1681 aggtttcctg gtcggcagtt cgggaatttc gttaaaccct cccgtttaga ggaaggagaa 1741 gtcgtaacaa ggtttccgta ggtgaacctg

a. b. c. Fig. 1.1 Growth habit of P. submarinum in liquid culture. (a) germlings at 2-3 months (b) mature tufts at 6-8 months (c) mature tufts after 2 years.

a. b. c.

Fig. 1.2 Scanning electron micrographs of Pseudendoclonium submarinum. (a) germling (b) upright filaments (c) individual tuft

Fig. 3.1 Maximum likelihood phylogram inferred from partial 18s rDNA sequences of 25 green algal taxa illustrating ordinal affiliation of four species of Pseudendoclonium as the genus is currently circumscribed. Horizontal branch lengths are proportional to the mean number of nucleotide substitions per site. Numbers above branches or adjacent to nodes reflect bootstrap support based on 2000 and 100 replications under MP and ML criteria respectively. Single numbers are shown where bootstrap support was less than 50 % under the MP criterion. Unnumbered branches received less than 50 % bootstrap support under both MP and ML optimality criteria.

Fig. 3.2 Maximum likelihood phylogram inferred from 18S rDNA sequences of 22 green algal taxa illustrating ordinal affiliation of three species of Pseudendoclonium as the genus is currently circumscribed. Horizontal branch lengths are proportional to the mean number of nucleotide substitions per site. Numbers above branches or adjacent to nodes reflect bootstrap support based on 2000 and 100 replications under the MP and ML criteria respectively. Single numbers are shown where bootstrap support was less than 50 % under the MP criterion. Unnumbered branches received less than 50 % bootstrap support under both MP and ML optimality criteria.

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